Economist and former finance minister Pena had vowed to continue recognising Taiwan, and his win erased Taipei's fear that Paraguay would ditch ties with the self-ruled island in favour of Beijing.
His challenger Efrain Alegre had mooted a shift in allegiance to China, which claims Taiwan as part of its territory and pushes to isolate it internationally.
During their 20-minute telephone call, Tsai congratulated Pena and thanked him for "expressing his firm stance to maintain and strengthen the Taiwan-Paraguay relations", her office said in a statement.
Pena told Tsai he looks forward to visiting Taiwan and meeting her "as soon as possible", and hopes the two allies can "continue to deepen exchanges and cooperation", her office said.
"I look forward to working with you to advance our bilateral cooperation & the well-being of our peoples," Tsai tweeted after the call.
Paraguay is one of Taiwan's few remaining allies in Latin America and the only one in South America -- after Panama, El Salvador, the Dominican Republic, Nicaragua and Honduras switched allegiance to China in recent years.
Latin America has been a key diplomatic battleground for China and Taiwan since the end of the Chinese Civil War in 1949.
Beijing has spent decades convincing Taipei's diplomatic allies to switch sides, gaining nine since Tsai took office in 2016.
Honduras made the latest switch to Beijing in March, leaving only 13 countries that diplomatically recognise Taipei.
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